Endotoxin enhances liver alcohol dehydrogenase by action through upstream stimulatory factor but not by nuclear factor-kappa B

Liver alcohol dehydrogenase (ADH) is increased by physiological stress and by chronic administration of growth hormone (GH). Endotoxin plays a role in the pathogenesis of alcoholic liver disease. The effect of lipopolysaccharide (LPS), the endotoxin component of Gram-negative bacteria, was determined on liver ADH. LPS given daily to rats for 3 days increased ADH mRNA, ADH protein, and ADH activity. Nuclear factor-kappaB (NF-kappaB) in the liver nuclear extracts bound to an oligonucleotide specifying region -226 to -194 of the ADH promoter, whereas upstream stimulatory factor (USF) was shown previously to bind to a more proximal site. LPS increased NF-kappaB and USF binding to the ADH promoter. The NF-kappaB (p65) and NF-kappaB (p50) expression vectors inhibited the transfected ADH promoter activity, which contrasts with the previously demonstrated stimulation by an USF expression vector. The binding activities of STAT5b and of C/EBPbeta, which mediate the effect of GH on ADH, were not changed or decreased, respectively, by LPS, indicating that GH plays no intermediary role in the effect of LPS. This study shows that LPS increases ADH and that this effect is mediated by increased binding of USF to the ADH promoter and not by NF-kappaB, which has an inhibitory action.     

The bidirectional upstream element of the adenovirus-2 major late promoter binds a single monomeric molecule of the upstream factor.

The adenovirus-2 major late promoter (Ad2MLP) upstream element (Ad2MLP-UE) contains a sequence of interrupted dyad symmetry. By inverting this element we have found that it functions in a bidirectional manner both in vivo and in vitro. Footprinting and binding kinetics studies have demonstrated that both orientations of the upstream element bind the sequence-specific upstream factor (UEF) in a similar fashion. These data strongly suggest that the dyad symmetric sequence is sufficient for fully functional binding of the UEF. Binding studies of the UEF to the Ad2MLP-UE indicate that, contrary to prokaryotic palindromic promoter elements which bind multimers of specific factors, the entire Ad2MLP dyad symmetric upstream element binds a single monomeric UEF molecule.     

Molecular evolution of mammalian class I alcohol dehydrogenase

Phylogenetic relationship and the rates of evolution of mammalian alcohol dehydrogenases (ADHs) have been studied by using the amino acid sequences from the human (ADH alpha, ADH beta, and ADH gamma), rat, mouse, and horse (ADH E and ADH S). With the maize ADH1 and ADH2 used as references, the patterns of the amino acid replacements in the beta-sheets, alpha-helices, and random coils in each of the catalytic and coenzyme-binding domains were analyzed separately. The phylogenetic trees based on the different sets of amino acid substitutions consistently showed that (1) multiple ADHs in human and horse have arisen after mammalian radiation, (2) the common ancestor of human ADHs alpha and beta diverged from the ancestor of ADH gamma first and the former two ADHs diverged from each other more recently, and (3) the human ADHs are more closely related to the rodent ADHs than to the horse ADHs. Furthermore, the estimated branch lengths showed that the rodent ADHs are evolving faster than the other ADHs. This difference in evolutionary rate between the two groups of organisms is explainable either in terms of the difference in the number of cell generations per year or in terms of reduction of functional constraints.     

The protein factor which binds to the upstream activating sequence of Saccharomyces cerevisiae ENO1 gene.

Using a gel retardation assay it was shown that the 87 bp DNA fragment (UAS87) containing the upstream activating sequence (UAS) of S. cerevisiae EN01 gene and a nuclear extract gave rise to three migration-retarded species specific to UAS87. Heat- or proteinase-treatment of the nuclear extract revealed that these species were protein-DNA complexes. The precise binding region of the protein identified by DNaseI protection analysis was found to include a CCAAACA sequence which forms a dyad-symmetrical structure. The amount of one of the three migration-retarded species significantly increased when cells were grown in medium containing a gluconeogenic carbon source. The introduction of pGCR8, a multicopy plasmid containing GCR1 gene, a regulatory gene controlling the expression of several glycolytic enzymes, showed no effect on the amount of three migration-retarded species.     

Enhancer-site downstream binding protein activity is enriched in rat tissues that express the class I alcohol dehydrogenase gene.

The activity of the rat class I alcohol dehydrogenase (ADH) is enriched in certain tissues including the liver, intestine and testis. The tissue-specific expression of the gene encoding ADH in the rat was studied and found to closely correlate with tissue isozymic activity. A factor designated enhancer-site downstream binding protein (EDBP) was recently identified in the rat liver and found to interact with the proximal promoter of the class I ADH gene. The distribution of EDBP in nuclear extracts obtained from various tissues was examined based on its sequence-specific DNA binding property and found to correlate with tissue ADH expression. These findings suggest that EDBP is potentially a positive regulatory factor which is involved in controlling the tissue-specific expression of the ADH gene.     

Type I transforming growth factor beta receptor binds to and activates phosphatidylinositol 3-kinase

We have examined the interaction of transforming growth factor (TGF)beta receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFbeta increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFbeta receptors (TbetaR) associated with p85, but the association of TbetaRII appeared to be constitutive. The interaction of TbetaRI with p85 was induced by treatment with TGFbeta. The receptor association with PI3 kinase was not direct as (35)S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TbetaRII blocked ligand-induced p85-TbetaRI association and PI3 kinase activity. In TbetaRI-null R1B cells, TGFbeta did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TbetaRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TbetaRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TbetaRI kinase inhibitor LY580276, suggesting a causal link between TbetaRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFbeta receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TbetaRI serine-threonine kinase can potently induce PI3 kinase activity.